Composition for gene introduction into cell

ABSTRACT

The present invention is directed to a composition for gene transfer which composition contains a quaternary ammonium salt represented by formula (1):                    
     wherein A represents                    
     (wherein each of R 1 , R 2 , R 3 , R 4  and R 5 , which are identical to or different from one another, represents a C9-C17 aliphatic group); X 1  represents a halogen atom; and n is an integer from 1 to 10 inclusive; and a method for introducing a gene into a cell by use of the composition. 
     The composition enables effective delivery and expression of a gene which previously could not be effectively expressed in cells due to the low ratio at which the gene is delivered into cells. Therefore, the composition is advantageously used as a gene transfer reagent or a pharmaceutical.

TECHNICAL FIELD

The present invention relates to a composition for gene transfer intocells, as well as to a method for gene transfer into cells by use of thecomposition.

BACKGROUND ART

Plasma membranes have low permeability to some compounds which are usedas drugs, and thus the drugs fail to exhibit sufficient intracellularpharmacological effect. Low plasma membrane permeability may beattributable to, for example, the compound having low lipid solubilityor high molecular weight. A typical example of such a compound to whichplasma membranes exhibit low permeability is a gene.

At present, therapeutic treatment by use of such drugs to which plasmamembranes exhibit low permeability, in particular, a gene, is conductedby way of injection, etc. However, the permeation of such a drug intothe inside of cells is so poor that it is unable to provide satisfactorytherapeutic effect.

To solve this problem, there have been proposed various conventionaltechniques known as drug delivery systems (DDS). As such systems thereare, for example, liposomes formed primarily of phospholipids, emulsionscomposed of surfactants and oils such as soybean oil, mixed micellesmade of lipids and surfactants, and microcapsules/microspheres made ofbiodegradable or non-degradable polymers. However, the conventionaltechniques have failed to increase permeability of a drug through themembrane; rather, in vitro evaluation has revealed that conventionaldrug delivery systems have an effect of decreasing cell-membranepermeability of a drug. This is because the drug itself is encapsulatedin the drug delivery system, and release of the drug from the systemserves as a determining factor. Despite this drawback, drug deliverysystems have been attracting close attention, and have been applied tomany drugs. This is because when a drug is encapsulated in a drugdelivery system, in vivo degradation of the drug can be suppressed, andin vivo kinetics of the drug can be controlled, thus eventuallyincreasing the in viva drug concentration in the vicinity of the targettissue or cells. Even in the case of liposomes, one of the typical drugdelivery systems, although suppression of drug degradation andcontrolling the in vivo kinetics can both be achieved with relativeease, particles of liposomes ultimately accumulate in the vicinity ofthe target tissue or cells at high concentration and release the drug,and the remainder of the drug delivery steps depend solely on thepermeability of the drug through the plasma membrane. Thus, althoughliposomes can attain an increased drug concentration near target cells,they have no effect on the permeability of the drug through the plasmamembrane.

In some in vitro cases, a drug delivery system increases the rate ofdrug delivery into cells. Example cases include use of phagocytic cellssuch as macrophages and monocytes. Phagocytic cells readily ingestmicroparticles, such as liposomes, by endocytosis, and therefore,transferability of the drug into cells may be increased if the drug isencapsulated in a drug delivery system rather than administered alone.In this case, permeability of the drug through the plasma membrane isnot increased. However, if the drug can be temporarily incorporated intocellular vesicles, such as endosomes and lysosomes, together with thedrug delivery system and happens to be stable in thesemicroenvironments, the drug can further enter the cytoplasm, resultingin increased drug transferability into cells.

Also, in recent years, extensive research has been directed to genetransfer into non-phagocytic cells, which is achieved through formationof a complex with a gene and cationic lipids (or liposomes containingthe cationic lipids) or through encapsulation of a gene into liposomescontaining the cationic lipids, thereby allowing the gene to beexpressed within the cells. Even though almost nothing is known aboutthe gene transfer mechanism into cells, reagents related to theabove-described research are widely commercialized (including reagentssuch as lipofectAMINE, lipofectACE, lipofectin, transfectam, andgenetransfer). Presently, biological researchers are using thesereagents on a daily basis as very useful tools for gene transfer intocells, and this method serves as a substitute for the virus andmicroinjection methods. However, these commercialized reagents have manydrawbacks as described in the following a) to d). a) These reagents, ascommercialized products, are not stable, and thus are not suitable forstorage. Many of these commercialized products are sold in the form of adispersion in water, and the pH of their aqueous solvents are usuallyvery low pH (for example, the pH is 3.5 for lipofectAMINE andlipofectACE, and 4.3 for lipofectin). Because of this low pH, lipidstend to degrade during storage. It has often been pointed out thatefficiencies of gene transfer into cells and of gene expression by useof liposomes, etc. do not have satisfactory reproducibility. One reasonfor this is the inherent instability of the products. (b) Anotherdrawback is that those products are very unstable in the presence offetal bovine serum added to a medium for cell culture. As a matter offact, the commercialized products employ the following protocol for genetransfer: Before gene transfer, the cultured medium containing fetalbovine serum is replaced with serum-free medium; and then, aftercompletion of the gene transfer, the serum-free medium is replaced byserum-containing medium. Recently, it has become clear that thesecommercialized products are also very unstable in blood as well as invivo. (c) A further drawback is that those commercialized products arenot suitably designed for easy handling. Many of the commercializedproducts, such as lipofectAMINE, lipofectACE, and lipofectin, areprovided in the form of a dispersion in water. For gene delivery,aqueous solvents that contain gene samples are added to these products.However, this protocol only allows the products to form complexes withthe genes where genes bind only to the outside of the liposomes.Therefore, these products cannot yield vesicles having the genesencapsulated inside the liposomes. (d) Moreover, a further drawback isvery strong cytotoxicity originated from those products. As is wellknown, the primary purpose for which biological researchers use thesecommercialized gene transfer reagents is to obtain the cells that havebeen transformed with exogenous genes and are capable of expressingthose genes, and to subsequently use the obtained cells in subsequentstudies. For such a purpose, in most cases, whether or not a minorportion of cell population dies during the gene transfer step isimmaterial, so long as such transformed cells can be obtained. Thus,there have been commercialized some reagents making use of cationiclipids alone or in combination with liposomes, for delivering into cellsa drug such as gene which in nature cannot be permeated through themembrane. However, those commercialized reagents involve many problems,as mentioned above. Thus, it is no exaggeration to say that applicationof such reagents to human use (such as gene therapy) is unthinkable atpresent. (Note: Gene therapy includes ex vivo and in vivo methods. Inthe case of ex vivo treatment, in which cells are taken out of apatient, treated in vitro, and subsequently returned to the patient,cytotoxicity raises a great problem.)

As pointed out above, it would be no exaggeration to conclude that thereis no conventional satisfactory method whereby a gene—which, except forspecial cases such as the case of phagocytic cells or some commerciallyavailable gene transfer reagents, is poorly permeated through the plasmamembrane, is poorly delivered into the inside of cells, or encountersdifficulty in manifesting its activity inside the cells—can be deliveredinto cells, after which the gene is allowed to exert its pharmacologicalefficacy.

Thus, an object of the present invention is to improve permeationthrough a plasma membrane, transmembrane delivery, and intracellularexpression of a gene, to which the plasma membrane has low permeability,in order to deliver the gene into the inside of cells, or to express thegene within the cells.

DISCLOSURE OF THE INVENTION

In view of the above, the present inventors have performed diligentstudies in order to solve the problems involved in delivery of a gene tothe inside of cells whose plasma membranes have low permeability to thegene, and further to improve expression of the gene inside the cell. Asa result, the inventors have found that administration of a genetogether with a quaternary ammonium salt represented by formula (1) tocells leads to efficient gene expression, not only in vitro but also invivo. The present invention has been achieved on the basis of thisfinding.

Accordingly, the present invention provides a composition for genetransfer into cells, which composition comprises a quaternary ammoniumsalt represented by the following formula (1):

wherein A represents

(wherein each of R¹, R², R³, R⁴, and R⁵, which are identical to ordifferent from one another, represents a C9-C17 aliphatic group); X¹represents a halogen atom; and n is an integer from 1 to 10 inclusive.

The present invention also provides a composition for gene transfer intocells, which composition comprises a quaternary ammonium saltrepresented by formula (1) and a gene.

The present invention also provides a gene transfer method comprisingapplying a composition containing a quaternary ammonium salt representedby formula (1) and a gene into a cell either in vivo or in vitro.

BEST MODE FOR CARRYING OUT THE INVENTION

In formula (1), which represents the quaternary ammonium salt to beincorporated into the composition of the present invention, examples ofC9-C17 aliphatic groups represented by R¹, R², R³, R⁴, and R⁵ includelinear or branched, saturated or unsaturated C9-C17 aliphatic groups.Among them, linear or branched C9-C17 alkyl groups are preferred, andlinear or branched C11-C15 alkyl groups are more preferred. Also, C9-C17linear alkyl groups are more preferred, with C11-C15 linear alkyl groupsbeing particularly preferred. Specifically, undecyl, tridecyl, andpentadecyl linear alkyl groups are particularly preferred. R¹, R², R³,R⁴, and R⁵ may be identical to or different from one another. However,identical groups are preferred from the point of view of manufacturing.

In formula (1), the halogen atom represented by X¹ is not particularlylimited. However, chlorine or bromine is preferred.

In formula (1), n represents an integer from 1 to 10 inclusive. Amongsuch integers, 1 and 10 are particularly preferred. When n is 1, A ispreferably

Also, when n is 10, A is preferably

In the composition for gene transfer according to the present invention,the amount of the quaternary ammonium salt represented by formula (1)varies in accordance with the gene employed, use of the composition, andthe physical form of the composition. Basically, any amount that allowsthe gene to be transferred into cells is sufficient. For example, theweight ratio of the composition to the gene is preferably 1:1 to 1:1000;more preferably 1:1-1:100.

Genes used in the present invention may be in the form of eitheroligonucleotides, DNA, or RNA. In particular, genes resulting intransformation upon in vitro gene transfer and those becoming activeupon in vivo gene expression are preferred. Examples for the latter caseof in vivo expression include those for gene therapy and breeding ofindustrial animals, such as domestic animals and animals forexperimental use. When genes used for gene therapy are incorporated intothe composition of the present invention, the composition serves as apharmaceutical composition. Examples of genes for such gene therapyinclude antisense oligonucleotides, antisense DNA, antisense RNA, andgenes encoding physiologically active proteins such as enzymes andcytokines. When genes encoding a certain enzyme are used, a substancethat exhibits pharmacological effect due to the action of the enzyme maybe used in combination with such genes. For example, tumors may betreated by first delivering a thymidine kinase gene in advance andcausing expression in vivo (in tumors), and subsequently administeredacyclovir can kill the tumors.

In order to improve the efficiency of gene transfer, the composition ofthe present invention may further contain phospholipids and/orcholesterol. Examples of phospholipids which may be contained includephosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelin,plasmalogen, and phosphatidic acid. These phospholipids may be usedsingly or in combination. Preferably, phosphatidylethanolamine andphosphatidylcholine are used singly or in combination of two or morespecies; and use of phosphatidylethanolamine is particularly preferred.Fatty acid residues of these phospholipids are not particularly limited.Preferable fatty acid residues include C12-C18 saturated or unsaturatedfatty acid residues; and the palmitoyl group, oleoyl group, stearoylgroup, and linoleyl group are most preferred. The amount of thephospholipid to be incorporated into the composition of the presentinvention is preferably 0-80%, more preferably 10-70%, particularlypreferably 25-70% based on the mole fraction. In the case ofcholesterol, the amount is preferably 0-70%, more preferably 10-60%,particularly preferably 20-50%, based on the mole fraction.

When the quaternary ammonium salt represented by formula (1) is usedtogether with the phospholipid and/or cholesterol, the efficiency ofgene transfer increases significantly as compared with the case of soleuse of the quaternary ammonium salt. Particularly, a remarkable increasein efficiency is observed when the phospholipid and the quaternaryammonium salt represented by formula (1) are used in combination. Also,in formulae), when A is a quaternary ammonium salt represented by

use in combination with phospholipid provides a remarkable increase inthe efficiency of gene transfer.

The amount of the quaternary ammonium salt to be incorporated into thecomposition of the present invention is, based on the mole fraction,preferably 5-100%, more preferably 10-75%, particularly preferably15-50%.

When the composition of the present invention contains the quaternaryammonium salt, and phospholipid or cholesterol, the mole ratio of thequaternary ammonium salt to the phospholipid or cholesterol ispreferably 1:9-9:1, more preferably 2:8-8:2, particularly preferably3:7-7:3. In this case, one type of phospholipid or a mixture of two ormore types of phospholipid can be used.

When the composition of the present invention contains the quaternaryammonium salt, phospholipid, and cholesterol, the mole ratio of themixture of the quaternary ammonium salt and phospholipid to thecholesterol is preferably 3:7-9:1, more preferably 4:6-9:1, particularlypreferably 5:5-8:2. In this case, one type of phospholipid or a mixtureof two or more types of phospholipid can be used.

Further, lipid-soluble vitamins such as vitamin E can be incorporatedinto the composition of the present invention.

With regard to the mode of the composition of the present invention, thequaternary ammonium salt (1) can be incorporated alone or can simply bemixed with the phospholipid and/or cholesterol. Also, in order to form aphospholipid membrane structure, the quaternary ammonium salt (1) can beincorporated alone or can be incorporated in combination with thephospholipid and/or cholesterol. Although no particular limitation isimposed on the modes and manufacturing methods for the lipid membranestructure, examples thereof include a dried lipid mixture, a lipidmixture dispersed in an aqueous solvent, a lipid mixture dispersed in anaqueous solvent and further dried, and a lipid mixture dispersed in anaqueous solvent and then frozen.

Concerning the method of manufacturing the dried lipid mixture, forexample, lipid components to be used for the mixture are first dissolvedwith an organic solvent such as chloroform, and subsequently subjectedto in vacuo drying by use of an evaporator or to spray-drying by use ofa spray-dryer.

With regard to the dispersion mode in an aqueous solvent, though noparticular limitation is imposed on the lipid membrane structure,examples thereof include multilamellar liposome, unilamellar liposome,O/W emulsion, W/O/W emulsion, spherical micelle, and string-shapedmicelle, as well as an amorphous multi-layered structure. Although noparticular limitation is imposed on the particle size of the lipidmembrane structure, the diameter of the liposomes and emulsions is 50 nmto several μm, and that of the spherical micelle is 5 nm to 50 nm.However, in the case in which the concept of a diameter cannot beapplied to the string-shaped micelle and amorphous multilayer structure,the concept of the thickness of one layer can be employed, which is 5 nmto 10 nm, and layers are stacked one on another, thus forming theamorphous multilayer structure.

No particular limitation is imposed on the aqueous solvent. However, inaddition to water, examples thereof include the following: sugarsolutions such as those containing glucose, lactose, or sucrose;polyalcohol solutions such as those containing glycerin or propyleneglycol; physiological saline; buffered solutions such asphosphate-buffered solutions, citrate-buffered solutions, andphosphate-buffered physiological saline; and media for cell culture. Inorder to effect long-term stable preservation of lipid membranestructure in the dispersion mode in an aqueous solvent, the followingpoints are important: from the physical viewpoint, such as prevention ofaggregation, electrolytes are eliminated from the aqueous solvent to thegreatest possible extent; and from the viewpoint of lipid chemicalstability, the pH of the aqueous solvent is set to a range from weakacidity to neutral (pH3.0-8.0) and dissolved oxygen is removed from thesolvent by means of bubbling with nitrogen. Further, use of sugarsolutions for preservation of lyophilized samples and spray-driedsamples, as well as use of sugar solutions and polyalcohol solutions forcryopreservation, can achieve effective preservation.

Although no particular limitation is imposed on the concentrations ofsuch aqueous solvents, sugar solutions preferably have concentrations of2-20% (W/V) more preferably 5-10% (W/V); polyalcohol solutionspreferably have concentrations of 1-5% (W/V), more preferably 2-2.5%(W/V); and buffered solutions preferably have concentrations of 5-50 mM,more preferably 10-20 mM.

Also, no particular limitation is imposed on concentrations of thelipids forming the lipid membrane structure in the aqueous solvent. Thetotal lipid concentration of the quaternary ammonium salt (1),phospholipid, and cholesterol used for the lipid membrane structure ispreferably 0.001 mM-100 mM, more preferably 0.01 mM-20 mM.

In order to manufacture the dispersion mode of the lipid membranestructure in the aqueous solvent, an aqueous solvent is added to theabove-described dried lipid mixture, followed by emulsification by useof an ultrasonic homogenizer, a high-pressure jet homogenizer, or anemulsifier such as a homogenizer. Alternatively, without use of suchdried lipid mixtures, a well-known liposome manufacturing method such asthe reverse-phase evaporation method can be used, and no particularlimitation is imposed on such manufacturing methods. In order to controlthe particle size, extrusion can be carried out under high pressurethrough a membrane filter having a uniform pore size.

Further, examples of the methods of drying the lipid membrane structuredispersed in an aqueous solvent include typical lyophilization and spraydrying. Concerning the aqueous solvent for this purpose, as mentionedabove, sugar solutions such as a sucrose or lactose water solution arepreferred. Among the merits for further drying the lipid membranestructure already manufactured, such dried forms allow long-termpreservation of the lipid membrane structure. Additionally, whengene-containing solutions are added to the dried forms, the lipidmembrane structure is efficiently rehydrated so that the gene is alsoeffectively retained by the lipids forming the lipid membrane structuresuch as liposomes.

A conventional method may be used for further freezing the dispersionmode of the lipid membrane structure in the aqueous solvent. Asdescribed above, this method preferably employs aqueous solvents such asa sugar solution or a polyalcohol solution. The merits of furtherfreezing the lipid membrane structure include permitting long-termpreservation of the lipid membrane structure.

The composition of the present invention which contains a gene(gene-containing composition) will next be described.

The mode of the gene-containing composition of the present invention maybe a mixture of quaternary ammonium salt (1) and a gene; a mixture ofquaternary ammonium salt (1), a gene, and phospholipid and/orcholesterol; a mixture of a gene and a lipid membrane structure formedof quaternary ammonium salt (1) alone or in combination withphospholipid and/or cholesterol; or a form in which a gene is carried onthe lipid membrane structure. Here, “carry” indicates that the gene isburied in the lipid membrane, present on the surface of the lipidmembrane, present inside the membrane, buried in a lipid layer, orpresent on the lipid layer.

As is the case with the lipid membrane structure, no particularlimitation is imposed on the form and production method of thegene-containing composition. For example, the composition may be formedinto a dry mixture, a dispersion in an aqueous solvent, or a dry orfrozen form of the dispersion.

The dry mixture of a lipid and a gene may be prepared by means of, forexample, dissolving a lipid component and a gene in an organic solventsuch as chloroform, and subsequently subjecting the solution to vacuumdrying by use of an evaporator or to spray-drying by use of a spraydryer.

Non-limiting examples of the dispersion of the mixture of a lipidmembrane structure and a gene in an aqueous solvent includemultilamellar liposome, unilamellar liposome, O/W emulsion, W/O/Wemulsion, spherical micelle, string-shaped micelle, or an amorphousmulti-layered structure. No particular limitation is imposed on theparticle size of the mixture, the composition of the aqueous solvent, orthe concentration of the mixture in the aqueous solvent.

Aqueous dispersions of a mixture of the lipid membrane structure and agene may be prepared through several methods having differentcharacteristic features, yielding different forms of the resultingmixtures of the lipid membrane structure and gene.

According to the first manufacturing method, an aqueous solvent is firstadded to the above-mentioned dry mixture of lipid and gene, followed byemulsification by use of a commonly-employed emulsifier such as ahomogenizer, ultrasonic emulsifier, or a high-pressure jet homogenizer.In order to control the particle size, extrusion may be carried outunder high pressure through use of a membrane filter having a uniformpore size. In this case, in order to prepare the dry mixture of lipidand gene, the gene must first be dissolved in an organic solvent. Thismethod is advantageous in that interaction between the gene and thelipid membrane structure can be fully utilized, so that the gene canenter the inside of the multi-layer when the lipid membrane structurehas a layered structure. Thus, in general, the method enables more genesto be carried by the lipid membrane structure.

According to the second manufacturing method, after lipid components aredissolved in an organic solvent, the organic solvent is removed so as toobtain a dry material. Then, an aqueous solvent that contains a gene isadded to the dry material, followed by emulsification. In order tocontrol the particle size, extrusion can be carried out under highpressure through a membrane filter having a uniform pore size. Thismethod can be applied to genes that are difficult to dissolve in anorganic solvent but are easily dissolved in an aqueous solvent. Thismethod is advantageous in that genes can be retained by the inneraqueous phase of liposomes.

According to the third manufacturing method, an aqueous solvent thatcontains a gene is added to lipid membrane structures (such asliposomes, emulsions, micelles, or layered structures) which has alreadybeen dispersed in another aqueous solvent. Therefore, this method isonly applied to water-soluble genes. Further, by this method, genes arelater added separately to the lipid membrane structure that has beenprepared in advance. Because of this, if the gene is large in size, itis unable to enter the inside of the lipid membrane structure and onlybinds to the surface thereof. When liposomes are used as the lipidmembrane structure, this third method is known to provide a sandwichstructure in which the gene is sandwiched by liposomal particles(generally called a complex). The merit to this third method is that thelipid membrane structure, such as that of the liposomes, emulsions,micelles, and layered structures, dispersed in an aqueous solvent can bestored after manufacturing, and can be used not only for one type ofgene but also commonly used for other types of gene. Further, when thismethod is used, the dispersion containing only the lipid membranestructure is prepared in advance. Because of this, there is no need toconsider degradation of drug during emulsification. Also, particle sizecan be easily controlled. Thus, this manufacturing method is more easilycarried out than are the first and second methods.

According to the fourth manufacturing method, a lipid membrane structureis first dispersed in an aqueous solvent, followed by drying. An aqueoussolvent that contains a gene is added to the dried material. As is thecase with the third manufacturing method, this method can be appliedonly to water-soluble genes. However, the third and fourth manufacturingmethods clearly differ in the state of the lipid membrane structure andgene. In the case of the fourth manufacturing method, first a lipidmembrane structure dispersed in an aqueous solvent is prepared, followedby drying to obtain dried materials. After this step, the lipid membranestructure exists in a solid state as fragments of the lipid membrane. Asmentioned above, obtaining such solid state lipid fragments requires useof an aqueous sugar solution, preferably a sucrose or lactose solution,serving as an aqueous solvent. When an aqueous solvent that contains agene is added to the solid state lipid fragments, they are quicklyhydrated to reconstitute the lipid membrane structure, as water isabsorbed. At this point, the resulting composition that retains thegenes inside the lipid membrane structure is generated. In contrast, inthe case of the third manufacturing method, when the gene is large insize, it is unable to enter the inside of the lipid membrane structureand only binds to its surface. One advantage of the fourth manufacturingmethod is that once the materials are manufactured, they can be used notonly for one type of gene but also commonly for other types of genes.Another advantage is that since the aqueous dispersion containing thelipid membrane structure alone is prepared in advance, degradation ofthe drug during emulsification is not a consideration. Further, theparticle size can be easily controlled. Thus, this manufacturing methodis more easily carried out than are the first and second methods. Inaddition, since the product is prepared by means of lyophilization orspray-drying, its storage stability is reliably ensured, enabling use asa commercial product; after rehydration of the dry product by use of agene-containing solution, the particle size is restored to the originalsize, and even the large gene can be easily retained inside the lipidmembrane structure.

Concerning other methods for preparing the dispersion mode of themixture of the lipid membrane structure and a gene, a well-known methodfor preparation of liposomes such as the reversed-phase evaporationmethod can be used. In order to control the particle size, extrusion canbe carried out under high pressure through a membrane filter having auniform pore size. Example methods of drying the above-mentioned mixtureof the lipid membrane structure and genes dispersed in the aqueoussolvent include lyophilization and spray drying. For this aqueoussolvent, as is the case with use of the lipid membrane structure alone,a sugar solution, preferably a sucrose or lactose solution, is used.

Conventional freezing methods may be used for freezing theabove-mentioned dispersed mixture of the lipid membrane structure andgene in an aqueous solvent. This aqueous solvent is preferably a sugaror polyalcohol solution, as is the case with the lipid membranestructure alone.

The composition of the present invention can be applied not only togenes but also to other drugs having very low lipid solubility andreagents which are difficult to be delivered into cells, such asphysiologically active peptides of high molecular weight and proteins.

By use of the composition of the present invention, genes can beefficiently transferred into cells either in vivo or in vitro. In thecase of in vitro transfer, genes can be delivered into target cells bymeans of adding the composition of the present invention to a suspensioncontaining target cells, or by culturing target cells with mediumcontaining the composition of the present invention. In the case of invivo transfer, the composition of the present invention can beadministered into a host. Administration can be carried out eitherorally or parentally. Oral administration may be carried out by use ofconventional formulations therefor, such as tablets, powders, andgranules. Parental administration may be carried out by use ofconventional formulations therefor, such as injection, instillation,ointments, and suppositories. Among these, parenteral administration ispreferred; particularly, injection is most preferred; and for itsadministration, intravenous injection or local injection at target cellsites or organs is preferred.

EXAMPLES

Next, the present invention will be described in detail by way ofexamples, which should not be construed as limiting the invention.

Example 1

Production of Empty Liposomes without a Gene

1-1. Production of Empty-liposomes Dispersion

Predetermined amounts of a quaternary ammonium salt, phospholipid, andcholesterol were dissolved in chloroform, and subsequently subjected tovacuum drying so as to obtain a lipid mixture. To the mixture, apredetermined amount of isotonic sucrose or lactose solution was added,and subsequently, while being warmed up, the mixture was subjected toemulsification by use of a homomixer. Thus, a crude liposomal dispersionwas obtained. Next, in order to adjust the particle size of theliposomes, the liposome solution was subjected to extrusion procedureunder high pressure through a membrane filter having a pore size of 0.22μm, to thereby obtain an empty-liposome dispersion.

1-2. Production of Lyophilized Empty Liposomes

A predetermined amount of the empty-liposome dispersion prepared in 1-1was aliquoted into vials, followed by lyophilization, to thereby obtainlyophilized liposomes.

Example 2

Production of Gene-containing Liposomes

2-1. Production of a Gene-containing Liposomal Dispersion (Type 1)

An empty-liposome dispersion (2 μmol/ml of the total lipidconcentration) manufactured in 1-1 was diluted with serum-free medium(D-MEM) to a concentration of 100 nmol/ml (the quaternary ammonium saltconcentration). Next, 100 μl of the empty-liposome dispersion, whichcontained 10 nmol of the quaternary ammonium salt, and 1 μg DNA (eitherPGV-C (luciferase gene) or pCAG-lacZ (β-galactosidase gene)) were mixedwith 100 μl D-MEM and left for 15 min; and then 0.8 ml serum-free D-MEMsupplemented with 12.5% FBS (the final concentration of FBS was 10%) wasadded to the mixture so as to obtain a 1-ml sample.

2-2. Production of a Gene-containing Liposomal Dispersion (Tupe 2)

The lyophilized dispersion of empty liposomes containing a concentrationequivalent to a 2 μmol/ml total lipid concentration, which wasmanufactured in 1-2, was rehydrated with distilled water to therebyreconstitute the original form (2 μmol/ml as a total lipidconcentration). Further, this solution was diluted with serum-freemedium (D-MEM) to 100 nmol/ml as a quaternary ammonium saltconcentration. Next, 100 μl of this liposomal dispersion containing a100 nmol equivalent quaternary ammonium salt and 1 μg DNA (PGV-C orpCAG-lacZ) was mixed with 100 μl serum-free medium (D-MEM), and left for15 min. Further, to the mixture, 0.8 ml D-MEM supplemented with 12.5%FBS (the final concentration of FBS was 10%) was added to obtain 1 ml ofsolution to thereby obtain a sample.

2-3. Production of a Gene-containing Liposome Dispersion (Tupe 3)

Distilled water that containing DNA (PGV-C or pCAG-lacZ) was added forrehydration (1 μg DNA/10 nmol of the quaternary ammonium salt) to thelyophilized empty liposomes manufactured in 1-2 (2 μmol/ml equivalent asthe total lipid concentration), and left for 15 min. Then the mixturewas diluted to a final concentration of 1 μg/ml DNA with D-MEMsupplemented with 10% FBS to thereby obtain a sample.

2-4. Production of a Gene-containing Liposomal Dispersion (Tupe 4)

The empty liposomal dispersion manufactured in 1-1 (2 μmol/ml as thetotal lipid concentration) was diluted to 400 nmol/ml as the quaternaryammonium salt with serum-free medium (D-MEM). Next, 500 μl of thisliposomal dispersion, containing 200 nmol of the quaternary ammoniumsalt, and 500 μl of serum-free medium (D-MEM) containing 20 μg DNA(pCAG-lacZ) were mixed, and left for 5 min., to thereby obtain a sample.

2-5. Production of a Gene-containing Liposomal Dispersion (Tupe 5)

To the lyophilized empty liposomes equivalent to 2 μmol/ml of the totallipid and manufactured in 1-2, distilled water was added for rehydrationto thereby reconstitute the original form (2 μmol/ml of the total lipidconcentration), and the solution was further diluted with serum-freemedium (D-MEM) so as to adjust the concentration to 400 nmol/ml as thequaternary ammonium salt. Next, 500 μl of this liposomal dispersion,containing 200 nmol of the quaternary ammonium salt, and 500 μl ofserum-free medium (D-MEM) containing 20 μg DNA (pCAG-lacZ) were mixedand left for 5 min, to thereby obtain a sample.

2-6. Production of a Gene-containing Liposomal Dispersion (Tupe 6)

To the lyophilized empty liposomes equivalent to 2 μmol/ml of the totallipid and manufactured in 1-2, distilled water containing DNA(pCAG-lacZ) was added (1 μg DNA/10 nmol of the quaternary ammonium salt)for rehydration, and left for 15 min. Further, this liposomal dispersionwas diluted to a final concentration of 20 μg/ml DNA with serum-freemedium (D-MEM), to thereby obtain a sample.

2-7. Production of a Gene-containing Liposomal Dispersion (Tupe 7)

pCAG-lacZ DNA contained in the sample of the gene-containing liposomaldispersion (type 5) manufactured in 2-5 was replaced with pCAG-TK (athymidine kinase gene). In all other respects, the type 7 sample wasmanufactured in the same manner as was the type 5 sample.

Test Example 1

Measurement of Luciferase Activity Respective types of tumor cells wereplated onto 6-well plates at a concentration of 1×10⁵-8×10⁵ cells/well,and cultured for 24 h in medium supplemented with 10% FBS, after whicheach well was washed once with serum-free medium. Then, 1 ml of theliposomal dispersion containing,PGV-C (see 2-1, 2-2, and 2-3; the finalconcentration was 1 μg DNA/10 nmol of the quaternary ammonium salt/ml)was added to each well, and reacted at 37° C. for 5 h. Then, after eachwell was washed with serum-free medium once, culture medium supplementedwith 10% FBS was added to each well; and, after cells were furthercultured for 2 days, luciferase assay was carried out.

Luciferase assay was performed as described below. Each well was washedtwice with phosphate-buffered saline (−) [PBS (−)]. Then, 150 μl of acell-solubilizing solution (LCβ) was added to each well, and the wellplates were left at room temperature for 15 min. Then the cells werescraped off from the plate substrate by use of a cell scraper. Eachlysate was subjected to centrifugation at 12,000 rpm for 2 min. Uponmixing 20 μl of the supernatant with 100 μl of a luminescent reagent,luminescence was measured by use of a lumi photometer (TD-4000,Laboscience). The amount of protein in each sample was estimated by useof BCA Protein Assay Reagent. Luciferase activity was expressed asemitted amount/mg protein. The results are shown in Tables 1 and 2.

TABLE 1 In vitro luciferase activity obtained through use of differentliposomes (D-MEM medium supplemented with 10% FBS) Method for Luciferaseactivity Composition of liposomal membrane preparing a (light units/mgprotein sec) [Cationic lipid appears first] liposomal Colo320 mEIILHEC-1A HRA ES-2 [Membrane composition props. dispersion (colon (uterus(uterus (ovary (ovary in Exs. are on the mole basis.] Remarks containinga gene cancer) cancer) cancer) cancer) cancer) Comp. CommercialCompositional lipid film⁷⁾ 489 838 3185 643 245 Ex. 1 Genetransfer prop.of the (Wako Pure Chemicals) membrane; 1) Comp. Commercial Compositionalcomplex⁸⁾ 176 3933  944 Ex. 2 LipofectACE prop. of the (LifeTechnologies) membrane; 2) Comp. Commercial Compositional Complex 429Ex. 3 LipofectAMINE prop. of the (Life Technologies) membrane; 3) Comp.Commercial LIPOFECTIN Compositional Complex  70 Ex. 4 (LifeTechnologies) prop. of the membrane; 4) Comp. Commercial DMRIE-CCompositional Complex 143 Ex. 5 (Life Technologies) prop. of themembrane; 5) Comp. Leaf Huang^(a)) Compositional Complex  8  6  0 Ex. 6prop. of the membrane; 6) Comp. SA^(b))/DOPE^(c))/DLPC^(d)) = 2/4/4 type3  8 194  0 Ex. 7 Comp. only DC-6-14^(e)) Cationic type 1  4  0 Ex. 8lipid along Ex. 1 DC-6-12/DOPE = 5/5 type 1 869 Ex. 2 DC-6-14/DOPE = 5/5type 1 2671  4877  1237  Ex. 3 DC-6-16/DOPE = 5/5 type 3 11823  1423 Ex. 4 DC-6-16/DOPE = 4/6 type 3 929 Ex. 5 DC-6-12/DOPE/Chol^(f)) = 4/3/3type 1 1244  323 Ex. 6 DC-6-14/DOPE/Chol = 4/3/3 type 1 3096  103503  71498  1682  Ex. 7 DC-6-14/DOPE/Chol = 4/3/3 type 2 71029  Ex. 8DC-6-16/DOPE/DLPC = 4/2/4 type 1 11930  Ex. 9 DC-6-14/DOPE/Chol =2.9/4.2/2.9 type 1 466 6111  1094  Ex. 10 DC-6-14/DOPE/Chol =3.6/3.6/2.8 type 1 2455  10776  1070  Ex. 11 DC-6-14/DOPE/Chol =4.6/1.8/3.6 type 1 1607  3161  421 Ex. 12 DC-6-14/DOPE/Chol = 5/2/3 type1 1111  3532  261 Ex. 13 DC-6-16/DOPE/DLPC = 3/4/3 type 2 469 629 Ex. 14DC-6-16/DOPE/DLPC = 4/4/2 type 2 423 1923  Ex. 15 DC-6-18:1/DOPE/DLPC =2/4/4 type 2 785 Ex. 16 DC-6-18:1/DOPE/Chol = 2/4/4 type 2 2951  prop.:proportion

a) See Biochem. Biophys. Res. Comm., vol. 179, No. 1, 280-285 (1991)

b) SA: Stearylamine (typical cationic lipid)

c) DOPE: Dioleylphosphatidylethanolamine

d) DLPC: Dilauroylphosphatidylcholine

e) DC-6-12:O,O′-N-didodecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride

DC-6-14:O,O′-N-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolaminechloride

DC-6-16:O,O′-N-dihexadecanoyl-N-(α-trimethylammonioacetyl)diethanolaminechloride

DC-6-18:1:O,O′-N-dioctadecenoyl-N-(α-trimethylammonioacetyl)diethanolaminechloride

f) Chol: cholesterol

1) N-[α-trimethylammonioacetyl]-didodecyl-D-glutamate/DOPE/DLPC=2/4/4(mole ratio)

2) Dimethyloctadecylammonium bromide/DOPE=2.9/7.1 (weight ratio)

3)2,3-Dioleyloxy-N-[2-(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneammoniumtrifluoroacetate/DOPE=3/1 (weight ratio)

4) N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride/DOPE=5/5 (weight ratio)

5) 1,2-Dimyristyloxypropyl-3-dimethylhydroxyethylammoniumbromide/cholesterol=1/1 (mole ratio)

6) 3-β-[N-(N′,N′-dimethylaminoethane)carbamoyl]-cholesterol/DOPE=6/4(mole ratio)

7) The commercial product used was a “lipid film.” A gene-containingsolution (aqueous solvent) was added to the “lipid film” so that 1 μgDNA was contained per 10 nmol of cationic lipid contained in thecomponent, followed by mixing in a vortex mixer, to thereby obtain agene-containing aqueous liposomal dispersion.

8) The commercial product and the method described in the literature: Toan aqueous dispersion of empty liposomea (or a lipid membranestructure), a gene-containing solution (aqueous solvent) was added sothat 1 μg DNA was contained per 10 nmol of cationic lipid contained inthe component, to thereby obtain a dispersion of gene-liposome complex.

TABLE 2 In vitro luciferase activity obtained through use of differentliposomes (D-MEM medium supplemented with 10% FBS) Method for Luciferaseactivity Composition of liposomal membrane preparing a (light units/mgprotein sec) [Cationic lipid appears first] liposomal Colo320 HEC-1A HRAKF [Membrane composition props. dispersion (colon (uterus (ovary (ovaryin Exs. are on the mole basis.] Remarks containing a gene cancer)cancer) cancer) cancer) Comp. Commercial Compositional lipid film⁷⁾ 4893185  643  23 Ex. 1 Genetransfer prop. of the (Wako Pure Chemicals)membrane; 1) Comp. Commercial Compositional complex⁸⁾ 176 3933  944 Ex.2 LipofectACE prop. of the (Life Technologies) membrane; 2) Comp.Commercial Compositional Complex 429 Ex. 3 LipofectAMINE prop. of the(Life Technologies) membrane; 3) Comp. Commercial LIPOFECTINCompositional Complex  70 Ex. 4 (Life Technologies) prop. of themembrane; 4) Comp. Commercial DMRIE-C Compositional Complex 143 Ex. 5(Life Technologies) prop. of the membrane; 5) Comp. Leaf Huang^(a))Compositional Complex  8  6  0 Ex. 6 prop. of the membrane; 6) Comp.SA^(b))/DOPE^(c))/DLPC^(d)) = 2/4/4 type 3  8 194  0 Ex. 7 Ex. 17TC-1-12^(g))/DOPE = 5/5 type 3 2887  342 Ex. 18 TC-1-12/DOPE/DLPC =2/4/4 type 1 8325  6245  Ex. 19 TC-1-12/DOPE/DLPC = 3/4/3 type 2 5429 Ex. 20 TC-1-12/DOPE/DLPC = 3/4/3 type 3 3459  1204   40 Ex. 21TC-1-12/DOPE/LysoPC^(h)) = 2/4/4 type 3 1598  Ex. 22 TC-1-12/DOPE/Chol =5/2.5/2.5 type 3 2176  Ex. 23 TC-1-12/DOPE/Chol/DLPC = 2/4/2/2 type 33909   89 Ex. 24 TC-1-12/DOPE = 4/6 type 2 1378  Ex. 25TC-1-12/DOPE/DPLC = 4/4/2 type 2 3355  446 648 Ex. 26 TC-1-12/DOPE/DPLC= 5/4/1 type 2 2897  414 488 Ex. 27 TC-1-12/DOPE/Lyso-LPC^(i)) = 2/4/4type 2 4499  Ex. 28 TC-1-12/DOPE/Lyso-MPC^(j)) = 2/4/4 type 2 3258  Ex.29 TC-1-12/DOPE/Chol = 2.5/2.5/5 type 2 1680  223 Ex. 30TC-1-12/DOPE/Chol = 2.5/5/2.5 type 2 1606  287 Ex. 31 TC-1-12/DOPE/Chol= 5/4.5/0.5 type 2 1221  prop.: proportion

a)-f) and 1)-8): See Table 1

g) TC-1-12: O,O′,O″-tridodecanoyl-N-({overscore(ω)}-trimethylammoniodecanoyl)-tris(hydroxymethyl)-aminomethane bromide

h) LysoPC: Lisophosphatidylcholine

i) Lyso-LPC: Lisolauroylphosphatidylcholine

j) Lyso-MPC: Lisomyristoylphosphatidylcholine

As is apparent from Tables 1 and 2, the gene transfer compositionaccording to the present invention exhibited enhanced gene transferability as compared with commercial gene transfer reagents.

Test Example 2

X-gal Staining

Each type of tumor cells were plated in amounts of 1×10⁵-8×10⁵ in asix-well plate, followed by culturing for 24 hours in a medium addedwith FBS (10%) and washing once with serum-free medium. Subsequently, 1ml of a gene-containing liposomal dispersion (gene: pCAG-lacZ) preparedin Example 2 (2-1, 2-2, or 2-3; final DNA concentration=1 μg/10 nmolquaternary ammonium salt/ml) was added to each well, and reaction wasallowed to proceed for five hours. When five hours have elapsed, thewells were washed once with serum-free medium. FBS (10%)-supplementedmedium was added and subjected to incubation for two days, and thenX-gal staining.

X-gal staining was performed as follows. Briefly, the sample was washedonce with PBS(−), then fixed for 3-4 minutes by use of PBS(−) containing1% formaldehyde, 0.2% glutaraldehyde, and 0.02% NP40, followed bywashing PBS(−) three times, each for 10 minutes. Ultimately, stainingwas performed for 5-8 hours at 37° C. by use of a mixture solutioncontaining 5 mM K₄[Fe(CN)₆], 5 mM K₃[Fe(CN)₆], 0.01% sodium deoxycholicacid, 0.02% NP40, 2 mM MgCl₂, and 0.1% X-gal. Under a microscope, cellswere counted at least 1,000 in number, to thereby obtain a LacZ-positive number. The results are shown in Tables 3 through 5.

TABLE 3 Percentage in vitro LacZ positive cells obtained through use ofdifferent liposomes (in medium supplemented with 10% FBS) Method forPercentage LacZ positive cells preparing a (count of positive cells per100 cells) Composition of liposomal membrane a liposomal HEC-1A mEIILHRA ES-2 SW626 KF KOC-3S [Cl appears first] dispersion con- (uterus(uterus (ovary (ovary (ovary (ovary (ovary [MCP in Exs. are on the molebasis.] Remarks taining a gene cancer) cancer) cancer) cancer) cancer)cancer) cancer) Comp. Commercial CP of lipid film⁴⁾ 2.6 7.6 10.1  8.69.2 3.3 6.0 Ex. 1 Genetransfer membrane; 1) Comp. Commercial CP ofcomplex⁵⁾ 0.6 4.7 3.3 0   2.4 Ex. 3 LipofectAMINE membrane; 2) Comp.Commercial CP Complex 4.0 1.3 10.1  0.6 0.5 Ex. 5 DMRIE-C membrane; 3)Ex. 5 DC-6-12^(a))/DOPE^(b))/Chol^(c)) = 4/3/3 type 1 21.2  10.6  38.9 24.7  14.8  14.5  4.8 Ex. 6 DC-6-14/DOPE/Chol = 4/3/3 type 1 42.1  23.7 16.0  5.9 Ex. 32 DC-6-12/DOPE/Chol = 4/3/3 type 3 8.7 8.4 9.3 13.7  Ex.33 DC-6-14/DOPE/Chol = 1.8/5.4/2.8 type 1 14.9  21.4  7.4 4.8 CL:Cationic lipid CP: Compositional proportion MCP: Membrane compositionalproportions

TABLE 4 Percentage in vitro LacZ positive cells obtained through use ofdifferent liposomes (in medium supplemented with 10% FBS) Method forPercentage LacZ positive cells preparing a (count of positive cells per100 cells) Composition of liposomal membrane a liposomal HEC-1A mEIILHRA ES-2 SW626 KF KOC-3S [Cl appears first] dispersion con- (ovary(ovary (ovary (ovary (ovary (ovary (ovary [MCP in Exs. are on the molebasis.] Remarks taining a gene cancer) cancer) cancer) cancer) cancer)cancer) cancer) Comp. Commercial CP of lipid film 6.0 5.4 0.9 6.6 31.3 8.5 1.4 Ex. 1 Genetransfer membrane; 1) Comp. Commercial CP of Complex0.1 0.6 1.0 0.2 0   0.4 0   Ex. 3 LipofectAMINE membrane; 2) Comp.Commercial CP Complex 0.1 1.0 0   0.2 0   4.5 0   Ex. 5 DMRIE-Cmembrane; 3) Ex. 5 DC-6-12^(a))/DOPE^(b))/Chol^(c)) = 4/3/3 type 1 7.48.6 8.3 Ex. 6 DC-6-14/DOPE/Chol = 4/3/3 type 1 6.5 11.5  12.3  Ex. 32DC-6-12/DOPE/Chol = 4/3/3 type 3 4.6 Ex. 33 DC-6-14/DOPE/Chol =1.8/5.4/2.8 type 1 4.4 9.1 22.2  7.5 CL: Cationic lipid CP:Compositional proportion MCP: Membrane compositional proportions

TABLE 5 Percentage in vitro LacZ positive cells obtained through use ofdifferent liposomes (in medium supplemented with 10% FBS) Method forPercentage LacZ positive cells Composition of liposomal membranepreparing a (count of positive cells per 100 cells) [CL appears first]liposomal dispersion HEC-1A COS-1 [MCP in Exs. are on the mole basis.]Remarks containing a gene (uterus cancer) (fibroblast cells) Comp.Commercial CP of lipid film  2.6 Ex. 1 Genetransfer membrane; 1) (WakoPure Chemicals) Comp. Commercial CP of Complex 0   Ex. 3 LipofectAMINEmembrane; 2) (Life Technologies) Ex. 20 TC-1-12^(a))/DOPE^(b))/DLPC^(d))= 3/4/3 type 3 11.5 Ex. 23 TC-1-12/DOPE/Chol^(c))/DLPC = 2/4/2/2 type 318.0 Ex. 34 TC-1-12/DOPE/DLPC = 2/4/4 type 3 14.1 Ex. 35TC-1-12/DOPE/Chol = 3/4/3 type 1 3.7 Ex. 36 TC-1-12/DOPE/Chol = 3/4/3type 3 11.6 Ex. 37 TC-1-12/DOPE/Chol/DLPC = 2/4/3/1 type 3 15.3 CL:Cationic lipid CP: Compositional proportion MCP: Membrane compositionalproportions

a) DC-6-12:O,O′-N-didodecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride

DC-6-14:O,O′-N-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolaminechloride

DC-6-16:O,O′-N-dihexadecanoyl-N-(α-trimethylammonioacetyl)diethanolaminechloride

TC-1-12: O,O′,O″-tridodecanoyl-N-({overscore(ω)}-trimethylammoniodecanoyl)-tris(hydroxymethyl)-aminomethane bromide

b) DOPE: Dioleylphosphatidylethanolamine

c) Chol: Cholesterol

d) DLPC: Dilauroylphosphatidylcholine

1) N-[α-trimethylammonioacetyl]-didodecyl-D-glutamate/DOPE/DLPC=2/4/4(mole ratio)

2)2,3-Dioleyloxy-N-[2-(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneammoniumtrifluoroacetate/DOPE=3/1 (weight ratio)

3) 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammoniumbromide/cholesterol=1/1 (mole ratio)

4) The commercial product used was a “lipid film.” A gene-containingsolution (aqueous solvent) was added to the “lipid film” so that 1 μgDNA was contained per 10 nmol of cationic lipid contained in thecomponent, followed by mixing in a vortex mixer, to thereby obtain agene-containing aqueous liposomal dispersion.

5) The commercial product and the method described in the literature: Toan aqueous dispersion of empty liposomes (or a lipid membranestructure), a gene-containing solution (aqueous solvent) was added sothat 1 μg DNA was contained per 10 nmol of cationic lipid contained inthe component, to thereby obtain a dispersion of gene-liposome complex.

As is apparent from Tables 3 to 5, the gene transfer compositionaccording to the present invention exhibited enhanced gene transferability as compared with commercial gene transfer reagents.

Test Example 3

X-gal Staining (2)

Each type of tumor cells were intraperitoneally inoculated to each nudemouse in amounts of 5×10⁶ (mEIIL, ES-2)-6×10⁷ (HRA). After one day (inthe case of HRA), about 10 days (in the case of ES-2), or about 3 weeks(in the case of mEIIL), 1 ml of the gene-containing liposomal dispersion(gene: pCAG-lacZ) described above (2-4, 2-5, or 2-6; final DNAconcentration=20 μg/200 nmol quaternary ammonium salt/ml) wasintraperitoneally administered to the mouse. On the following day (inthe case of mEIIL and HRA) or two days later (in the case of ES-2),tumor cells were collected, and 3×10⁵-5×10⁵ cells were plated in thewells of a 6-well plate. The cells were incubated for 24 hours by use ofa medium supplemented with 10% FBS, followed by X-gal staining. X-galstaining was performed in a manner similar to that in Test Example 2.The results are shown in Table 6.

TABLE 6 Percentage in vivo LacZ positive cells obtained through use ofdifferent liposomes Percentage LacZ positive cells Method for (count ofpositive cells per 100 cells) Composition of liposomal membranepreparing a HRA mEIIL ES-2 [CL appears first] liposomal dispersion(ovary (uterus (ovary [MCP in Exs. are on the mole basis.] Remarkscontaining a gene cancer) cancer) cancer) Comp. Commercial GenetransferCP of Lipid film⁶⁾ 0.95 0.25 0.25 Ex. 1 membrane; 1) Comp. CommercialLipofectACE CP of complex⁷⁾ 0.62 Ex. 2 membrane; 2) Comp. CommercialLipofectAMINE CP of Complex 0.23 Ex. 3 membrane; 3) Comp. CommercialLIPOFECTIN CP of Complex 0.38 Ex. 4 membrane; 4) Comp. CommercialDMRIE-C CP of Complex 1.52 Ex. 5 membrane; 5) Ex. 38DC-6-12^(a))/DOPE^(b)) = 5/5 type 4 5.50 Ex. 39 DC-6-14/DOPE = 5/5 type5 1.08 Ex. 40 DC-6-14/DOPE = 5/5 type 6 1.04 Ex. 41DC-6-12/DOPE/Chol^(c)) = 4/3/3 type 4 4.71 1.29 2.43 Ex. 42DC-6-14/DOPE/Chol = 4/3/3 type 4 1.32 Ex. 43 DC-6-14/DOPE/Chol = 4/3/3type 6 0.96 Ex. 44 DC-6-14/DOPE/Chol = 1.8/5.4/2.8 type 4 4.32 Ex. 45DC-6-14/DOPE = 4/6 type 4 6.30 CL: Cationic lipid CP: Compositionalproportion MCP: Membrane composition proportions

a) DC-6-12:O,O′-N-didodecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride

DC-6-14:O,O′-N-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolaminechloride

b) DOPE: Dioleylphosphatidylethanolamine

c) Chol: cholesterol

1) N-[α-trimethylammonioacetyl]-didodecyl-D-glutamate/DOPE/DLPC=2/4/4(mole ratio)

2) Dimethyldioctadecylammonium bromide/DOPE=2.9/7.1 (weight ratio)

3)2,3-Dioleyloxy-N-[2-(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneammoniumtrifluoroacetate/DOPE=3/1(weight ratio)

4) N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride/DOPE=5/5 (weight ratio)

5) 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammoniumbromide/cholesterol=1/1 (mole ratio)

6) The commercial product used was a “lipid film.” A gene-containingsolution (aqueous solvent) was added to the “lipid film” so that 1 μgDNA was contained per 10 nmol of cationic lipid contained in thecomponent, followed by mixing in a vortex mixer, to thereby obtain agene-containing aqueous liposomal dispersion.

7) The commercial product and the method described in the literature: Toan aqueous dispersion of empty liposomes (or a lipid membranestructure), a gene-containing solution (aqueous solvent) was added sothat 1 μg DNA was contained per 10 nmol of cationic lipid contained inthe component, to thereby obtain a dispersion of gene-liposome complex.

As is apparent from Table 6, the gene transfer composition according tothe present invention exhibited enhanced gene transfer ability ascompared with commercial gene transfer reagents.

Test Example 4

Life-prolonging Effect in Tumor-bearing Mice

Each type of tumor cells were intraperitoneally inoculated to nude micein amounts of 3×10⁵ (HRA), 1×10⁶ (mES-2), or 5×10⁶ (day 0). From day 7(in cases of HRA and ES-2) or from day 10 (in the case of mEIIL), 1 mlof the gene-containing liposomal dispersion (gene: pCAG-TK) describedabove (2-7; final DNA concentration=20 μg/200 nmol quaternary ammoniumsalt/ml) was intraperitoneally administered to each mouse. For each of13 consecutive days starting from day 9, i.e., until day 21 (in cases ofHRA and ES-2), or for each of 13 consecutive days starting from day 12,i.e., until day 24 (in the case of mEIIL), acyclovir wasintraperitoneally administered to each mouse twice a day at a dose of 35mg/kg. To mice of a control group, pCAG-lacZ gene was administered inplace of pCAG-TK. The significance test employed was the Cox-Mantelmethod. The results are shown in Table 7.

TABLE 7 Life-prolonging effect of liposomes in a peritoneum-implantedmodel Count of Days of Survived mice Composition of liposomal membraneimplanted Number Days of 50% at the end Significance [mole ratio] GeneOncocyte cells of mice observation Survival of observation test Ex. 46DC-6-14^(a))/DOPE^(b)) = 5/2 pCAG-TK HRA 3 × 10⁵ 12 70 40 5 p < 0.05(C.G.) DC-6-14/DOPE = 5/2 pCAG-lacZ HRA 3 × 10⁵ 12 70 34 1 Ex. 47DC-6-14/DOPE = 5/2 pCAG-TK mEIIL 5 × 10⁶  8 85 85 6 p < 0.05 or more(C.G.) DC-6-14/DOPE = 5/2 pCAG-lacZ mEIIL 5 × 10⁶  8 85 67 1 Ex. 48DC-6-14/DOPE/Chol^(c)) = 1.8/5.4/2.8 pCAG-TK mEIIL 5 × 10⁶  8 85 85 5 p< 0.05 or more (C.G.) DC-6-14/DOPE/Chol = 1.8/5.4/2.8 pCAG-lacZ mEIIL 5× 10⁶  8 85 60 2 Ex. 49 DC-6-12/DOPE/Chol = 4/3/3 pCAG-TK ES-2 1 × 10⁶12 77 50 3 p < 0.05 (C.G.) DC-6-12/DOPE/Chol = 4/3/3 pCAG-lacZ ES-2 1 ×10⁶ 12 77 37 0 C.G.: Control group

a) DC-6-12:O,O′-N-didodecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride

DC-6-14:O,O′-N-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolaminechloride

b) DOPE: Dioleylphosphatidylethanolamine

c) Chol: cholesterol

As is apparent from Table 7, the gene transfer composition according tothe present invention exhibited excellent life-prolonging effect.

INDUSTRIAL APPLICABILITY

The composition of the present invention enables effective delivery andexpression of a gene which previously could not be effectively expressedin a cell due to the low ratio at which the gene is introduced intocells. Therefore, the composition is advantageously used as a genetransfer reagent or a pharmaceutical.

What is claimed is:
 1. A composition for delivering genes or proteinsinto cells, which comprisesO,O′-ditetradecanoyl-(α-trimethylammonioacetyl)diethanolamine halide,one or more phospholipids, and cholesterol.
 2. A composition accordingto claim 1, wherein the one or more phospholipids are selected from thegroup consisting of phosphatidylethanolamine, phosphatidylcholine,phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,cardiolipin, sphingomyelin, plasmalogen, and phosphatidic acid.
 3. Acomposition according to claim 1, wherein the one or more phospholipidsare selected from the group consisting of phosphatidylethanolamine andphosphatidylcholine.
 4. A composition according to claim 1, wherein themole ratio of a mixture ofO,O′-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine halideand the one or more phospholipids to cholesterol is in the range of3:7-9:1.
 5. A composition according to claim 1, which forms liposomes.6. A composition according to claim 1, which further comprises at leastone gene, physiologically active polypeptide, or protein.
 7. A methodfor delivering genes, physiologically active polypeptides, or proteinsinto cells, comprising applying a composition as recited in claim 6 tothe cells in vitro.
 8. A composition according to claim 2, wherein themole ratio of a mixture ofO,O′-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine halideand the one or more phospholipids to cholesterol is in the range of3:7-9:1.
 9. A composition according to claim 3, wherein the mole ratioof a mixture ofO,O′-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine halideand the one or more phospholipids to cholesterol is in the range of3:7-9:1.
 10. A composition according to claim 2, which forms liposomes.11. A composition according to claim 3, which forms liposomes.
 12. Acomposition according to claim 4, which forms liposomes.
 13. Acomposition according to claim 2, which further comprises at least onegene or protein.
 14. A composition according to claim 3, which furthercomprises at least one gene or protein.
 15. A composition according toclaim 4, which further comprises at least one gene or protein.
 16. Acomposition according to claim 5, which further comprises at least onegene or protein.
 17. A composition according to claim 6, which furthercomprises at least one gene.
 18. A composition according to claim 6,which further comprises at least one or protein.
 19. A method fordelivering genes or proteins into cells, comprising applying acomposition as recited in claim 13 to the cells in vitro.
 20. A methodfor delivering genes or proteins into cells, comprising applying acomposition as recited in claim 14 to the cells in vitro.
 21. A methodfor delivering genes or proteins into cells, comprising applying acomposition as recited in claim 15 to the cells in vitro.
 22. A methodfor delivering genes or proteins into cells, comprising applying acomposition as recited in claim 16 to the cells in vitro.
 23. A methodfor delivering genes into cells, comprising applying a composition asrecited in claim 17 to the cells in vitro.
 24. A method for deliveringor proteins into cells, comprising applying a composition as recited inclaim 18 to the cells in vitro.
 25. A method for delivering or proteinsinto cells, comprising applying a composition as recited in claim 18 tothe cells in vivo.
 26. A method according to claim 25, wherein the oneor more phospholipids are selected from the group consisting ofphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelin,plasmalogen, and phosphatidic acid.
 27. A method according to claim 25,wherein the one or more phospholipids are selected from the groupconsisting of phosphatidylethanolamine and phosphatidylcholine.
 28. Amethod according to claim 25, wherein the mole ratio of a mixture ofO,O′-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine halideand the one or more phospholipids to cholesterol is 3:7-9:1.
 29. Amethod according to claim 25, which forms liposomes.
 30. A methodaccording to claim 25, wherein the composition contains at least one.31. A method according to claim 25, wherein the composition contains atleast one physiologically active protein.